Recognition of pathogens by the innate immune system activates TLR-mediated pathways, resulting in NF[unreadable]B induced transcription of inflammatory cytokines. These molecules subsequently direct the initiation of appropriate adaptive responses, leading ultimately to clearance or containment of the invading pathogen. Due to their potent biological activity, however, inappropriately prolonged or excessive release of pro-inflammatory mediators can result in deleterious effects for the host. This is exemplified by the acute phase cytokine interleukin-6 (IL-6), whose role in the development of the potentially pathogenic Th17 subset of T cells has recently been described. Given the dual nature of pro-inflammatory cytokines, essential for host defense but potentially lethal in excessive quantities, the mammalian immune system has evolved numerous regulatory mechanisms to precisely control the magnitude and time-course of inflammatory responses. The molecular mechanisms used in fine-tuning this process are therefore the subject of active investigation. Here we propose using a forward genetic analysis of inbred (C57BL/6J) and wild derived (MOLF/Ei, Czech/EiII, MSM/Ms) mouse strains that secrete different amounts of IL-6 in response to TLR stimulation as a novel approach to characterizing the regulation of early cytokine release. Initial studies from a panel of backcross mice have identified two loci on chromosomes 6 and 9 that make major contributions to the trait. Further, these two loci have a significant epistatic interaction (p <10-6, LRS 48.1). Our previous mapping combined with micro-array analysis had revealed the identity of one gene as Simpl (a.k.a. IRAK1BP1) with a negative contribution to the phenotype. We now propose to map via meiotic recombination and identify other gene(s), which positively contribute to the phenotype. We also propose to establish the mechanistic basic for their observed epistatic interaction. With respect to anti-inflammatory role of SIMPL, we propose to investigate the mechanism of inhibitory effect of SIMPL in TLR-mediated signaling. This proposal aims to approach the problem through more refined genetic analysis as well as biochemical and molecular characterization of the candidate genes. We believe that we will reveal important novel mechanisms of TLR signaling.